Notes: Promega T4 Polynucleotide Kinase was used to end-label an oligo representing a NF-kB binding sequence element with [γ-32P] ATP (7,000 Ci/mmol). Specific primers to IFN-β and IκB-α messenger RNA were used in RT-PCR to generate products for cloning into the pGEM®-T Easy Vector. The resulting plasmids were linearized with Nco I and Spe I, respectively, and used as templates for in vitro transcription using the Riboprobe® System to generate probes for use in an RNase protection assay. The antisense probes were labeled with [α-32P]CTP (800 Ci/mmol) in the Riboprobe® reactions. RNase ONE™ Ribonuclease was also used in this study. (3197)

Notes: T4 Polynucleotide Kinase was used to phosphorylate primers before ligating them into a phagemid vector to allow cloning of Sfi I-restricted DNA fragments. Phagemid was prepared from XL1-Blue cells using the Wizard® Minipreps DNA Purification System. Inserts were PCR amplified from the phagemid and purified using the Wizard® PCR Preps DNA Purification System. In separate experiments, the cDNA for the melanin concentrating hormone receptor 1 cDNA was cloned from total melanocyte RNA using MMLV Reverse Transcriptase. In vitro translation of the cDNA was performed using the TnT T7 Coupled Reticulocyte Lysate System and Canine Pancreatic Microsomal Membranes. (2602)

Notes: Total RNA was isolated from canine left ventricle muscle with the RNAgents® Total RNA Isolation System. The RNA was used for RT-PCR. T4 Polynucleotide Kinase was used to 5´-end label an oligo to generate a probe for cDNA library screening. (0912)

Notes: Ribozymes designed to cleave TNF-α cDNA were cloned into the pGEM®-3Z Vector before beingtranscribed in vitro. The transcribed ribozymes were then mixed with Transfectam® Reagent and injected into mice intraperitoneally. Tissues and cells isolated from the mice were assayed at later time points for fluor or radiolabled ribozymes. In vitro kinetic assays were also performed with ribozymes and RNA substrates labeled with T4 Polynucleotide Kinase. (2834)

Notes: Reporter studies were performed in the ME-180 squamous cell carcinoma cell line. One microgram of the firefly luciferase construct was contransfected with 1ng of pRL-CMV Vector (1,000:1 ratio of firefly reporter to Renilla control), and luciferase activities were measured with the Dual-Luciferase® Reporter Assay System. The Kanamycin Control RNA was used as a control template for primer extension analysis. (1065)